LEISHMANIA RNA VIRUS 1-MEDIATED CAP-INDEPENDENT TRANSLATION

Recently, a group of related Leishmania RNA viruses (Leishmania RNA vi rus 1 [LRV1]) has been isolated from Leishmania guyanensis and L. bras iliensis. These viruses persist in the cytoplasm and contain double-st randed RNA genomes. Miniexon sequences are absent from the 5' end of t he viral RNA, and the 5' end df the viral RNA lacks a cap structure, s uggesting that LRV1 has evolved a cap-independent mechanism of transla tion. Cap-independent translation of picornavirus genomic RNA requires a cis element, within the 5' untranslated region (UTR), referred to a s an internal ribosome entry site (IRES). In order to find out if the 5' UTR of LRV1 possessed IRES activity, we modified a Leishmania expre ssion vector, pX63NEO-GUS, so that it would produce a dicistronic tran script in which the neomycin phosphotransferase gene was separated fro m the downstream beta-glucuronidase (GUS) gene by the LRV1 5' UTR. Hig h levels of GUS activity were detected in L. major stably transformed with this plasmid. Elimination of the first 120 nucleotides of the vir al 5' UTR lowered GUS activity 10-fold, Furthermore, when the entire 5 ' UTR was eliminated, GUS activity was undetectable. These results, to gether with the absence of trans-spliced GUS transcripts, are consiste nt with the hypothesis that the 5' UTR of LRV1 functions as an IRES el ement. The ability to couple expression of genes via an IRES element s hould prove useful in genetic experiments with Leishmania spp.